The present invention relates generally to the production of chimeric molecules. In particular, it relates to the production of chimeric molecules (immunoligands) comprising at least a portion of a ligand molecule linked to an immunoglobulin constant region.
The basic immunoglobulin structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminal portion of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The C-terminal portion of each chain defines a constant region primarily responsible for effector function.
Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
Within the light and heavy chain, units made up of about 110 amino acids form discrete domains. Each domain is held together by a single internal disulfide bond. The heavy chain typically contains 4 such domains, while the light chain contains 2. The first N-terminal domain of the heavy chain, V.sub.H, interacts with the N-terminal domain of the light chain, V.sub.L, to produce the binding region of the antibody. Moving towards the C-terminus, the next three domains of the heavy chain are designated C.sub.H 1, C.sub.H 2, and C.sub.H 3, respectively. The mu and epsilon heavy chains contain an additional domain, C.sub.H 4.
Most heavy chains have a hinge region between the C.sub.H 1 and C.sub.H 2 domains consisting of a small number of amino acids. The hinge is flexible and allows the binding region to move freely relative to the rest of the molecule. At the hinge region are the disulfide bridges which hold the two dimers together, creating the tetramer structural unit.
The hinge region is the point on the molecule most susceptible to the action of protease. Treatment with the protease papain splits the molecule into three fragments, two of which are designated F.sub.ab fragments, and the other, the F.sub.c fragment. The F.sub.ab fragments each consist of an antigen binding domain and a C.sub.H 1 domain. Further proteolytic digestion of the F.sub.ab fragments releases the F.sub.v fragment, which consists only of the variable region.
The F.sub.c fragment, which consists of the C.sub.H 2 and C.sub.H 3 domains, is the portion of the immunoglobulin molecule that mediates effector functions. Depending upon the heavy chain in the immunoglobulin, a variety of effector functions are present. These include complement fixation, mediation of antibody dependent cell toxicity, stimulation of B cells, transport across the placenta, and longer serum half-life (see, generally, Hood, et al. Immunology (1984, 2d ed.), which is incorporated herein by reference).
Recent advances in molecular biology have provided methods for genetically engineering immunoglobulin molecules. While significant success has been made in changing the specificity of immunoglobulins (e.g., by replacing the complementarity determining regions), the majority of efforts seem to have focused on the constant region. For instance, the constant region has been totally or partly replaced with cytotoxic molecules to produce immunotoxins. Enzymes and other molecules have been attached to F.sub.v regions to produce chimeric molecules having novel properties. In addition, portions of immunoglobulins from different species have been combined to produce novel molecules. Recently, additional chimeric molecules have also been constructed composed of a T cell surface receptor glycoprotein (CD4) in the immunoglobulin gene superfamily fused to an immunoglobulin constant region.
While these modifications have proved successful in broadening the potential use of immunoglobulins, some significant limitations remain. For instance, although mammals create an immunoglobulin array with an astounding range of reactivity, producing an immunoglobulin with a precisely predetermined specificity is essentially always a matter of chance. Typically, a large number of candidates must be laboriously screened before a possibly suitable immunoglobuiin is identified. In many cases, an immunoglobulin having the desired specificity and affinity is never located, particularly if immunogenicity is also an important consideration. Problems of cross-reactivity and relatively weak binding affinity can especially hinder the usefulness of immunoglobulins in therapeutic applications.
Thus, there exists a need for increasing the specificity and improving binding affinity of immunoglobulins beyond the immunoglobulin gene superfamily, while retaining their other useful characteristics. The molecules should be capable of production in an economical manner, preferably suitable for pharmaceutical application. The present invention fulfills these and other needs.